The proposed research is to integrate MUSICAL with chip-based nanoscopy system for completely bypassing the need of fluorescence blinking and consequently avoiding problems of photo-chemical toxicity without compromising spatio-temporal resolution. This innovation is possible because MUSICAL uses fluctuations of intensity, howsoever induced (blinking or otherwise), for generating super-resolved images. Complementary to this, waveguide chip made of high-refractive index material (n = 2) generates dynamically varying speckle-like illumination patterns with spatial frequencies higher than what can be achieved using far-field diffraction limited optics. This illumination will induce fluorescence intensity fluctuations needed by MUSICAL. The innovation will result into controlled system-based imaging, instead of less-controllable blinking-based imaging. Further, it will allow significantly large field-of-view (~mm2) with resolution of (150 – 200 nm) using low NA (0.2) collection objective lens because the spatial frequencies of chip based illumination remain the same irrespective of the collection optics .
MUSICAL requires a threshold parameter that is provided by the user heuristically. Using a pattern recognition approach, the threshold can now be determined by Auto Threshold MUSICAL algorithm automatically. This makes MUSICAL more user-friendly and easy to use.
The link to Auto Threshold MUSICAL and a new nanoscopy dataset with results of autothreshold MUSICAL will be made available here after the publication of the results. This result is an outcome of the master thesis project of Sebastian Acuna.
MUSICAL is a live cell friendly fluorescence nanoscopy technique supporting resolution upto 35 nm. Use your widefield microscope to obtain super-resolved image streams of live cells.
The salient properties of MUSICAL are:
- Requires low power in comparison to most super-resolution techniques, therefore it is less phototoxic and is especially well-suited for live cells.
- Requires very few image frames (50 – 200 are sufficient in most cases), therefore suitable for dynamic systems such as live cells.
- Compatible with any dye or fluorescent protein in theory. Tested on Alexa dyes, GFP, RFP, YFP, CMP, SirTubulin, SirActin, MitoTracker dyes, etc.
- Compatible with dense or sparse samples and uses natural fluctuations in fluorescent intensity. Tested for cells and tissues without using any special imaging buffer (i.e. redox solutions).
- Tested on a variety of cameras, objective lenses (0.4 NA 20X to 1.49NA 100X oil immersion), and multi-channel acquisitions (4 channels so far).
- Works with TIRF and epifluorescence x-y-t image stacks.
It’s available in an ImageJ repository as an easy-to-install and easy-to-use plugin.